Work carried out in previous project periods has demonstrated that the combination of NMR spectroscopy, X-ray crystallography, and quantum chemical calculations serves as a highly productive approach to understanding sequence-structure-function relationships in iron-sulfur proteins. NMR spectra contain exquisitely sensitive information about electron nuclear interactions. This information, which is unavailable from X-ray crystal structures, provides insights into the chemical properties of the iron centers, details of their geometry, strengths of hydrogen bonds, and patterns of electron delocalization. NMR thus serves as a window for viewing the properties of the cluster that control redox potentials and regulate pathways of electron transfer. We have shown that high-level quantum chemical approaches can successfully correlate experimental NMR observables with geometric structures provided by high-resolution X-ray crystallography, and thus provide detailed insight to the factors tuning the properties of the metal site. In turn, NMR data and quantum calculations can provide structural constraints in cases where X-ray crystallography has not been feasible at sufficient resolution. We propose to use this coordinated approach to investigate a series of 1Fe and [2Fe-2S] proteins that are model systems in the study of electron transfer, with the aim of answering a number of questions regarding their structure-function relationships. The proteins to be studied include: a series of clostridial rubredoxins (monomeric 1Fe proteins) that have altered redox potentials, desulforedoxin (dimeric 1Fe protein), and four prototypical [2Fe-2S] proteins that have distinct spectral and functional properties (Anabaena vegetative ferredoxin, human ferredoxin, a Rieske protein, and Aquifex aeolicus ferredoxin). We propose, not only to collect and analyze NMR data, but also to collaborate with others to obtain crystal structures and electron-nuclear double resonance (ENDOR) data. An X-ray structure for reduced rubredoxin recently became available, but currently no structures are available for any reduced [2Fe-2S] proteins. Effort will be expended toward improving the calculations for 1Fe proteins and extending the methodology to [2Fe-2S] proteins. Effort will also be expended toward developing new methodology for paramagnetic NMR, in particular, for systems that are not amenable to established approaches.